Liposome formulations

ABSTRACT

The present invention relates to pharmaceutical formulations comprising an anti angiogenic compound such as a monoclonal antibody or fragment thereof selected from, for example, ranibizumab, which is a vascular endothelial growth factor binder which inhibits the action of VEGF, and a delivery agent selected from a pharmaceutically acceptable liposome. The formulations are useful in the treatment of a variety of angiogenic disorders and diseases in animals and people, and, preferably, in ophthalmic disorders selected from age-related macular degeneration, diabetic macular edema and corneal neovascularization.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims priority to U.S. Provisional PatentApplications 61/691,455, 61/791,693, and 61/862,300, filed Aug. 21,2012; Mar. 15, 2013; and Aug. 5, 2013, respectively, all of which areincorporated by reference herein in their entirety.

FIELD OF THE INVENTION

The present invention relates to pharmaceutical formulations comprisingan ophthalmic medication typically applied by intravitreal injection tothe eye and a liposomal deliver agent that permits topical applicationto the eye of said intravitreal medication. The present invention alsorelates to an anti-angiogenic compound such as a monoclonal antibody orfragment thereof selected from, for example, ranibizumab, which is avascular endothelial growth factor binder which inhibits the action ofVEGF, and/or a multi-kinase VEGFR and PDGFR inhibitor, for example,sunitinib, and a delivery agent selected from a pharmaceuticallyacceptable liposome. The formulations are useful in the treatment of avariety of angiogenic disorders and diseases in animals and people, and,preferably, in ophthalmic disorders selected from age-related maculardegeneration, diabetic macular edema and corneal neovascularization.

BACKGROUND OF THE INVENTION

Ophthalmic disease treatment typically requires either topicaladministration or intravitreal injection of the particular drug to theeye depending upon the particular disease or condition and theeffectiveness of the route of administration with respect to theparticular drug and disease. In certain diseases or conditions of theeye effective treatment can only be achieved if the drug is administeredby intravitreal injection. There are a litany of diseases and conditionsof the eye that are effectively treated by intravitreal injection. Atthe same time, these injections can cause and/or are associated withserious side effects including eye infections (endophthalmitis), eyeinflammation, retinal detachments and increases in eye pressure. Becauseof these side effects or risks topical treatment of the eye has beenboth the preferred route of administration of drugs to treat eyeconditions and the Holy Grail because in almost all cases, topicaladministration of the drug does not effectively treat certain eveconditions, especially those conditions that occur in the back of theeye. Thus there is a need to develop formulations that effectively treatsaid conditions and that eliminate the need to have intravitrealinjections. The present inventors believe they have found such avehicle, U.S. Pat. No. 6,884,879 discloses various anti-VEGF antibodies.This patent specifically describes and claims the monoclonal antibodyranibizumab which is approved and marketed under the brand nameLUCENTIS®. The antibodies disclosed therein are described as beingcapable of preventing, reversing and/or alleviating the symptoms ofvarious diseases and are described as having the ability to inhibitVEGF-induced proliferation of endothelial cells and the ability toinhibit VEGF-induced angiogenesis. LUCENTIS® is approved for neovascular(wet) age-related macular degeneration (AMD) at a dosage strength of 0.5mg (0.05 mL) by intravitreal injection one a month and, though lesseffective, the approved treatment may be administered to an injectionevery three months after an initial regimen of once a month for at leastfour months. LUCENTIS® is also approved in the United States for macularedema following retinal vein occlusion (RVO) using 0.5 mg (0.05 mL) on aonce-a-month intravitreal regimen. In addition, LUCENTIS® is approved inEurope and in the United States for diabetic macular edema in theinjectable formulation.

Sunitinib (SU-11248, Sutent), was approved January 2006 by FDA as amonotheraphy for the treatment of metastatic renal cell cancer andgastrointestinal stromal tumors. Sunitinib inhibits at least eightreceptor protein-tyrosine kinases including vascular endothelial growthfactor receptors 1-3 (VEGFR1-VEGFR3), platelet-derived growth factorreceptors (PDGFRα and PDGFRβ). This compound inhibits angiogenesis bydiminishing signaling through VEGFR1, VEGFR2, and PDGFRβ. PDGFRβ isfound in pericytes that surround capillary endothelial cells ¹(Roskosiki2007, PDF document attached). There is evidence that the combined use ofan anti-PDGF is superior to ranibizumab monotherapy in the treatment ofsome VEGF related diseases.

There are multiple side effects or potential side effects includingpatient discomfort associated with the intravitreal injection ofanti-VEGF antibodies and other ophthalmic drugs. The intravitrealinjection procedure requires a dedicated clean room with ordinaryaseptic rules; resuscitation facilities must be immediately available.Complications of this procedure include infectious endophthalmitis;retinal detachment and traumatic cataract. Other possible complicationsof intravitreal injection include intraocular pressure changes,especially intraocular pressure elevations. Injection relatedintraocular pressure elevations which can occur immediately afterinjection of any kind of medication and drug specific-relatedintraocular pressure changes which may be detected days or even monthsafter the injection. See Semin. Ophthamol. 2009 March-April;24(2):100-5.

There is an urgent unmet medical need for new topical treatment regimensof such anti-VEGF antibodies and other effective ophthalmic drugs suchas antimicrobials, antivirals, corticosteroids and anti-vascularendothelial growth factor agents which are the main classes of drugsthat are administered through intravitreal injections. The presentinvention comprises a combination of said Liposomes and any of saiddrugs within said drug classes in a topical formulation. While U.S. Pat.No. 6,884,879 generally discloses various possible delivery methods orreagents including liposomes and various mutes of administrationincluding topical administration of such VEGF monoclonal antibodies, theonly approved form of ranibizumab is the intravitreal form in a liquidformulation. There is a need for topical ranibizumab formulations thatare effective in treating people having VEGF related disorders includingophthalmic disorders.

The inventors have met this unmet need and have surprisingly found thatcertain liposomal formulations comprising such VEGF monoclonalantibodies and certain liposomes provide effective relief in patientshaving diabetic macular edema. The formulations can be effectivelyadministered topically to the affected eye and are more effective thantopical application of the intravitreal formulation. U.S. Pat. No.6,958,160 discloses and claims self-forming, thermodynamically stableliposomes. U.S. Pat. Pub. No. 2010/0076209, hereby incorporated byreference, discloses PEG-lipid conjugates for liposomes and drugdelivery. Various lipid based products including such self-formingthermodynamically stable liposomes marketed under the brand name Qsomes™(hereinafter Qsome) are sold by Biozone Laboratories for multipletherapeutic uses and via various routes of administration including bytopical administration to the skin. The inventors have discovered that apharmaceutical composition comprising ranibizumab and such self forming,thermodynamically stable liposomes and/or PEG-lipid conjugates can bedelivered topically to the eye of a patient in need of treatment of VEGFrelated ophthalmic diseases and conditions. The present inventionfurther comprises a topical formulation comprising a Qsome as recitedherein (self-forming thermodynamically stable liposome) and a drugwherein the formulation is suitable for the treatment of a posteriorsegment ophthalmic disease and/or a disease that is a combinationanterior segment/posterior segment disease. The claimed formulations aresuitable for topical administration and are particularly useful intreating ophthalmic diseases and conditions that are typically treatedvia a periocular route or via intravitreal administration (intraoculardelivery). Periocular administration includes subconjunctival, subtenon,retrobulbar and peribulbar administration. There are some drugs thathave been disclosed as being able to be delivered to the posteriorsegment by topical administration-these include ESBA105, ananti-INF-alpha single chain antibody; dexamethasone; nepafenac;memantine HCl; dorzolamide: brimonidine and betaxolol. It is believedthat topical administration of these drugs in a Qsome formulation willresult in more effective topical delivery and more effective posteriorsegment delivery. Preferably, however, the claimed invention comprises atopical formulation including a Qsome liposome as described herein anddrugs which heretofore have not been effectively delivered throughtopical administration.

Drugs typically given by intravitreal injection include antimicrobials,antivirals, corticosteroids and anti-vascular endothelial growth factoragents. The present invention comprises a liposomal formulationcomprising a self-forming thermodynamically stable liposome and anactive pharmaceutical agent selected from any one of or a combination ofan antimicrobial, antiviral, corticosteroid and anti-vascularendothelial growth factor agent wherein said formulation is suitable fortopical delivery to the eye of a patient to treat an ophthalmic diseaseor condition. Diclofenac, gatifloxacin, sparfloxcain lactate, GCV,demeclocycline, flubiprofen, doxorubicin, celecoxib, budesonide andcisplatin have been formulated in colloidal dosage forms fortranscorneal or transcleral delivery. Cationic liposomes containingpenicillin G, tropicamide and acetazolamide have been used to providemaximum drug, transport across the cornea relative to anionic andneutral liposomes. See Schaeffer et al. Liposomes in topical drugdeliver. Invest. Ophthalmol. Vis Sci. 198:222(2):220-7; Nagarsenker etal. Preparation and evaluation of liposomal formulations of tropicamidefor ocular delivery. Int J Pharma. 1999:190(1)163-71 and Hathout et al.Liposome as an ocular delivery system for acetazolamide: in vitro and invivo studies. AAPS Pharma Sci Tech. 2007; 8(1):1.

Diabetic retinopathy (DR) is the most common micro-vascular complicationof diabetes ²(Fong, 2004) and is the leading cause of new cases ofvision loss among working-aged adults. Diabetic macular edema (DME) isthe most common cause of vision loss in patients having DR. Theprevalence of DME is 3% recent diagnosis with about 75,000 new cases ofDME each year (USA). The number of worldwide patients having diabetes isa staggering 285 million. DME results from a series of biochemical andcellular changes that ultimately cause progressive leakage andexudation, leading to thickening of the retina and hard exudates within1 disc diameter of the center of the macula. DME is one of the mostcommon causes of impaired vision in patients with diabetes ³(Bhagat,2009). Approximately 50% of patients experience a loss of ≥2 lines ofBCVA after two years of follow-up ⁴(Meyer, 2007).

In DME, damaged blood vessels leak fluid into the central portion of theretina (macula) which leads to swelling. The macula is involved withsharp central vision. The fovea is at the center of the macula. DME canoccur in patients having type 1 or type 2 diabetes. Approximately 26million people in the United States have diabetes and 1.9 million newcases are diagnosed in people aged 20 and older each year. Up to 75,000new cases of DME are estimated to develop each year. DME is a leadingcause of blindness among the working-age population in most developedcountries. First line therapy for DME is laser surgery which seals theleaky blood vessels to diminish the leakage of fluid and reduce theamount of fluid in the retina. There is thus a significant need tocreate new formulations that provide therapeutic relief to thesepatients.

SUMMARY OF THE INVENTION

The present invention is a pharmaceutical formulation comprising ananti-angiogenic compound or other ophthalmic active ingredient and aliposome. The preferred anti-angiogenic compounds are anti-VEGFantibodies. The pharmaceutical formulation is preferably administered asa topical formulation to the eye of a patient in need of treatmentthereof. The pharmaceutical formulation is useful in the treatment ofVEGF related ophthalmic disorders including, for example, age relatedmacular degeneration and diabetic macular edema. The present inventionis also directed to formulations suitable for topical administration andwhich are effective in treating corneal neovascular disease.

The preferred anti-VEGF antibody is selected from ranibizumab(LUCENTIS®) and may be prepared as described in U.S. Pat. No. 6,884,879which is hereby incorporated-by-reference. This product is sold as aprescription intravitreal liquid formulation. Ranibizumab is arecombinant humanized IgG1 kappa isotype monoclonal antibody fragment.This antibody binds to and inhibits VEGF-A and has a molecular weight ofapproximately 48 kilodaltons. The product is produced in an E. coliexpression system in a nutrient medium that contains tetracycline. Theprescription product is supplied in a single use glass vial containing0.05 mL of a 10 mg/mL of ranibizumab. Other anti-VEGF antibodies such asbevacizumab may also be used to treat certain diseases and conditionssuch as corneal neovascular disease. The topical liposomal formulationdisclosed herein facilitates penetration of whole antibodies such asbevacizumab into eyes having abnormal neovascularature.

The liposomes useful in the present invention preferably comprise thoseliposomes that are described in U.S. Pat. No. 6,958,160 which is herebyincorporated by reference in its entirety. As described therein,liposomes are self-closed colloidal particles wherein membranes composedof one or more lipid bilayers encapsulate a fraction of the aqueoussolution in which they are suspended. As also recited in the '160patent, not all liposomes are the same and, in fact, liposomes can haveproblems including colloidal instability and manufacturing issues due toextreme conditions such as elevated pressures and temperatures as wellas high shear conditions—all of which can degrade the lipid components.Other issues associated with liposomes in general can includeheterogeneous distributions of sizes and numbers of bilayers which cancause or acerbate scale-up issues. In addition, sterilization conditionscan also create issues with liposomes. Liposomes also may have colloidalinstability due to aggregation while in suspension. This leads to fusionissues and the solution to this problem is typically lyophilizationwhich is a costly extra step. The liposomes disclosed in the '160 patenthave overcome these problems and it has been surprisingly found thatthese particular liposomes are effective when combined with anti-VEGFantibodies such as ranibizumab.

In particular, a formulation comprising self-forming, thermodynamicallystable liposomes and an anti-VEGF antibody is particular suitable fortopical application in the treatment of VEGF related ophthalmic diseasesand conditions.

The liposomes useful in the present formulation comprisediacylglycerol-PEG compounds. The melting point of these compounds isbelow about 40° C. and the acyl chains are greater than or equal toabout 14 carbons in length. These compounds are prepared as recited inthe '160 patent. The preferred lipid PEG conjugate is PEG-12-GDM(Polyethylene glycol 12-Glycerol Dimyristate). PEG stabilizes theliposomes by creating a steric barrier at the outer surface of theliposomes, the PEG chain has a molecular weight between about 300Daltons and 5000 Daltons. Liposome preparation entails merely mixing thelipid with an aqueous solution. These kinds of liposomes exist in thelowest energy state that the lipid can exist in while in aqueoussolution, reproducibility of liposome formation is no problem.

A defined lipid, lipid mixture, or lipids/compound mixture will formsimilar liposomes every time when mixed with the same aqueous solution.Above critical concentrations (around 20% weight to volume)non-liposomal structures will begin to form in aqueous solution. Theseliposomes exist in their lowest energy state and are thermodynamicallystable, self-forming liposomes. PEG-12 GDM forms very small vesicles sothey can be sterile filtered. Kinetic energy, such as shaking orvortexing, may be provided to the lipid solution and the aqueoussolution. The present formulation may also use other lipid-PEGconjugates as generally or specifically described in the U.S. Pat. No.6,958,160. In addition, other lipid PEG self-forming, thermodynamicallystable conjugates may also he used including those compounds describedin US Pat Pub. No, 2010/0076209. Table 1 below describes certain PEG-12GDM characteristics.

TABLE 1 PEG-12 GDM CHARACTERISTICS: SPONTA- SPONTA- MELTING NEOUS NEOUSPOINT LIPOSOMES LIPOSOMES LIPID (° C.) Pa Pv AT 20° C. AT 37° C. PEG-12Fluid @ 25 0.829 0.869 YES YES GDM MOLECULAR WEIGHT: 1068 g/mol OPTIMUMpH: 5-7 SOLUBILITY: Soluble in organic solvents.

In the ranibizumab topical formulation of the present invention. 1%weight volume of PEG-12 GDM based upon the final volume of solution wasused and having a pH of the topical solution of 5.5 and in theappropriate range of working with the liposome.

In addition to the anti-angiogenic compound (e.g. the anti-VEGFantibody) and the thermodynamically stable, self-forming liposome, theformulation may further comprise additional pharmaceutically acceptableexcipients. The preferred excipients are selected from the groupconsisting of excipients suitable for topical administration to the eye.These include surfactants, buffer reagents, pH modifiers, salts andother such ingredients.

The formulations are useful in treating VEGF related diseases andconditions and/or other angiogenic conditions. The present inventionthus includes use of such formulations to treat age related maculardegeneration, diabetic retinal diseases including diabetic macular edemaand corneal neovascularization. In a preferred embodiment, the inventioncomprises a topical formulation and encompasses a method of treatingsuch VEGF related diseases and conditions by topically administeringsuch formulations to the eye of a patient in need of treatment thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will he described in the following figures.

FIG. 1 shows data from a single patient treated with a liposomalranibizumab formulation having improved central foveal thicknessmeasurements using optical coherence topography (OCT-CFT) over an eightweek period after being dosed six drops per day for two weeks. At weekeight the patient showed an increase in CFT and treatment wasreinitiated at 10 weeks at a daily dose of 2 drops per day. FIG. 1 showsimprovement in CFT occurring again from the 12 week to 14 week period.

FIG. 2 shows the same clinical patient treated with a liposomalranibizumab formulation having improved visual acuity measurements(ETDRS BCVA) over an eight week period after being dosed six drops perday for two weeks. At week eight the patient showed a decrease in VA andtreatment was reinitiated at 10 weeks at a daily dose of 2 drops perday. FIG. 1 shows improvement in VA occurring again from the 12 week to14 week period.

FIG. 3 shows microscopy pictures of multilamellar liposomes andunilamellar liposomes.

FIG. 4 shows OCT results of Central Foveal thickness of patients treatedwith the liposomal formulation of ranbizumab over time.

FIG. 5 shows OCT results of Central Foveal thickness of the patients'contra lateral eyes over time.

FIG. 6 shows the results of BCVA over time for the study eyes in thethree patients.

FIG. 7 shows the results of BCVA over time for the contra-lateral eyes.

DETAILED DESCRIPTION

The present invention relates to pharmaceutical formulations and usesthereof wherein the preferred formulation comprises an anti-VEGFantibody and a self-forming, thermodynamically stable liposome. Thepresent invention also relates to a topical formulation comprising ananti-VEGF antibody and a self-forming, thermodynamically stableliposome. The invention further comprises a method of treating a VEGFrelated disease or condition comprising administration of a formulationcomprising an anti-VEGF antibody and a self-forming, thermodynamicallystable liposome to a patient in need of treatment thereof. In apreferred embodiment the VEGF related disease or condition is selectedfrom a diabetic retinopathy (DR).

While liposomes “in general” have been described in connection with thedelivery of various active ingredients, the art does not disclose orteach the combination of self-forming, thermodynamically stableliposomes in combination with anti-VEGF antibodies. The liposomes of theformulation are particularly suitable for delivery of anti-VEGFantibodies to a patient in need of treatment of a VEGF-related diseaseor condition and, in particular, to ophthalmic diseases or conditions.The liposomal formulations of the present invention are particularlysuited for topical administration to the eye of a patient in need oftreatment of, for example, diabetic macular edema or age-related maculardegeneration or corneal neovascularization. The liposomes of the presentinvention have desired fundamental properties that make them especiallysuitable for these topical formulations. The liposome suspensions arethermodynamically stable at the temperature of formulation. Thecompositions of the lipids that make up the liposome have severalfundamental properties. The lipids have packing parameters that allowthe formation of liposomes. The lipids include, as part of the headgroup, a polyethyleneglycol (PEG) or polymer of similar properties thatsterically stabilizes the liposomes in suspension. In addition, theliposomes have a melting temperature that allows them to be in liquidform when mixed with water or an aqueous solution.

As described in the U.S. Pat. No. 6,610,322, little or no energy need beadded when forming the liposomal suspensions in aqueous solution. In thepresent invention, the preferred method involves forming the liposomalsuspension in the presence of an aqueous solution containing the activeingredient-the anti-VEGF antibody. Self-assembly thus preferably occurswith the active ingredient rather than before the active ingredient isadded to the suspension. The lipid molecules disperse and self assembleinto the natural low energy state. The liposomes form large or smallunilamellar vesicles (SUVs) or multilamellar vesicles (MLVs) (see FIG.3) and as described in the Biozone Laboratories website for Qusomes™.

The PEG chain preferably has a molecular weight of between about 300Daltons and 5000 Daltons. Examples of suitable lipids include PEG-12 GDO(glycerol dioleate) and PEG-12 GDM (glycerol dimyristate). PEG-12 GDM isfluid at 25° C. and has packing parameters P_(a) and P_(v) of 0.853 and0.889 respectively. Each of these lipids form spontaneous liposomes at20° C., 37° C. and 60° C. The Pa may range between 0.84 and 0.88 and thePv between about 0.88 and 0.93. Preferably, the suitable compounds formliposomes instead of, for example, micelles. In addition, the lipidcomposition should have a phase transition temperature of between about0° C. and 100° C.—the lipid composition has a melting temperature whichallows the composition to be in liquid form when mixed with an aqueoussolution. Also, the bending elastic modulus of the composition should besuch that the lipid composition can form liposomes in an aqueousenvironment without the need for any or any significant energy input.Kinetic energy may be applied to the solution. The preferred bendingelastic modulus is between about 0 kt and 15 kt. The bending elasticmodulus is largely determined by the backbone and glycerol is apreferred backbone of the present invention although any equivalentbackbone in terms of bending elastic modulus and suitable functionalitymay also be used. The relative percentage by weight of the lipid in thefinal solution may range from greater than 0 to about 20 wt percent(w/w). The range may be between about 1% and 15 wt % or between about 1%and 10% or between about 1% and 5% wt/wt or between greater than 0% and4% wt/wt.

Mixtures of other molecules and lipids having PEG chains longer than 12may also be used in the present invention provided they form liposomes.For example, a mixture of PEG-45 GUS (glycerol distearate) andcholesterol forms liposomes. As described in the '322 patent, one ofordinary skill in the art can vary the variables including PEG chainlength and components to prepare a thermodynamically stable, freeforming liposome and such are included within the scope of the presentinvention and when combined with an anti-VEGF antibody. The amount ofcholesterol when added to the lipid before liposomal formation is up toabout 10% w/w.

In addition to the lipids described in the '322 patent, other lipids mayalso be utilized in the present invention. U.S. Pat. Pub. No.2010/0076209 describes certain PEG-LIPID conjugates that form liposomessuitable for drug delivery of specifically described active ingredients.There is no teaching of or reference to the delivery of anti-VEGFantibodies in the reference. In particular, diacylglycerol-polyethyleneglycol compound as described in the '209 publication may be utilized incombination with anti-VEGF antibodies in the formulations of the presentinvention. The general structure of the lipid compounds is shown in the'209 publication and includes compounds having the formula(R2)(R1)Glycerol-X-PEG-R1 and/or R1-PEG-X-Glycerol (R2)(R1) wherein R1is preferably either —OH or —OCH3; R2 and R3 are fatty acids including,and not limited to laurate, oleate, myristate, palmitate, stearate andlinoleate; and X represents a single linker or replicate linkers orcombination of two or more linkers in between the lipid and PEG. R2 andR3 may be the same or different. If R2 is at the C1 position ofglycerol, R3 can be located at either C2 or C3. The general structureincludes all racemers and structural isomers and/or functionalequivalents thereof.

R1 may also be selected from, for example, —NH2, —COOH, —OCH2CH3,—OCH2CH2CH3, —OCH2CH2OH, —COCH═CH2, —OCH2CH2NH2, —OSO2CH3, —OCH2C6H6,—OCH2COCH2CH2COONC4H4O2, —CH2CH2═CH2 and —OC6H6. Also R1 may be afunctional group that helps link or links therapeutic or targeting,agents to the surface of a liposome. These may include amino alkylesters, maleimide, diglycidyl ether, maleinimido propioinate, methylcarbamate, tosyhydrazone salts, azide, propargyl-amine, propargylalcohol, NHS esters, hydrazide, succinimidyl ester, succinimidyltartrate, succinimidyl succinate, and toluesulfonate salts. The presentinvention includes liposomal formulations having anti-VEGF antibodieswithin the liposomal composition and may further include any othertherapeutic compound including the anti-VEGF antibodies covalently boundor linked to the liposome via the R1 functional group. In such instance,the invention would include or be a combination formulation or aformulation having both a non-covalently bound active ingredient and acovalently bound active ingredient wherein said active may be the sameor different.

The linkers useful or suitable in this type of liposomal formulationinclude those described in the '209 publication and which is herebyincorporated by reference. Table 1 therein describes or lists suchsuitable linkers and which include amino, succinylamino acetamido,2-aminopentanamido, 2(2′)-R′-aminoacetyl etc. In each case, the lipidsform spontaneous liposomes at 20 and 37° C. as shown in Table 4 of the'209 publication. The present invention thus includes those lipidsdescribed as PEG-12-N1 -GDO; PEG-23-N2-GDO; PEG-18-N3-GDO;PEG-23-N4-GDO; PEG-8-S1-GDO; PEG-18-S2-GDO; PEG-12-S3-GDO;PEG-18-Ac1-GDO; PEG-12-Ac2-GDO; PEG-12-N1-GDM; PEG-12-N1-GDLO;PEG-12-S3-GDM; PeG-12-S3-GDLO; PEG-12-Ac2-GDM; PEG-12-Ac2-GDLO;PEG-23-N1-GDL; PEG-12kN1-GDP; PEG-23-Ac2-GDL and PEG-12-Ac2-GDP. GDLOmeans glycerol dilinoleate and GDP means glycerol dipalmitate. Each ofthe compounds are fluid at 25° C. and have packing parameters Pa rangingfrom 0.830 to 0.869 and Pv ranging from 0.872 to 0.924.

The present invention further includes known lipids that meet thephysical requirements recited herein and which, for example, are liquidat 25° C. and are self-forming at both 20° C. and 37° C. and havefunctionally equivalent or equivalent packing parameters and formthermodynamically stable liposomes with little or no energy input whencombined, with an aqueous solution.

The anti-VEGF antibodies useful in the present formulation include anyknown anti-VEGF antibody. These antibodies include whole antibodies orantibody fragments provided they have the requisite anti-VEGF biologicalproperties. In a preferred embodiment, the antibody is an antibodyfragment having the requisite anti-VEGF biological and pharmacologicalproperties. U.S. Pat. No. 6,884,879 discloses anti-VEGF antibodiesuseful in the present invention. Such antibodies include humanizedanti-VEGF antibodies and anti-VEGF antibody variants with propertiesthat include strong binding affinity for VEGF; the ability to inhibitVEGF promoted proliferation of endothelial cells and the ability toinhibit VEGF induced angiogenesis. The preferred binding affinity (Kd)is no more than about 5×10⁻⁹M and with an ED50 value of no more thanabout 5 nM for inhibiting VEGF-induced proliferation of endothelialcells in vitro. The antibodies include those that inhibit at least about50% tumor growth in an A673 in vivo tumor model at an antibody dose of 5mgs/kg. The most preferred antibody is sold under the brand nameLUCENTIS® (ranibizumab) and is approved for the treatment of age-relatedmacular degeneration and various forms of macular edema as anintravitreal formulation. The term “anti-VEGF antibody” includes wholeantibodies as well as antibody fragments. The range of diseases that canbe treated with anti-VEGF antibodies includes those diseases orconditions that are associated with angiogenesis or pathologicalangiogenesis conditions. These include cancer as well as intraocularneovascular syndromes such as proliferative retinopathies or age-relatedmacular degeneration (AMD), rheumatoid arthritis and psoriasis. Whilethe preferred route of administration is topical treatment to the eyeand the preferred disease modality is diabetic macular edema, theformulation recited herein may also be useful in other delivery modes(i.e., injectable intravenous infusion) and in the treatment of thelitany of VEGF related diseases and conditions.

The anti-VEGF antibodies include those produced from an isolated nucleicacid encoding a humanized variant of a parent anti-VEGF antibody whichparent antibody comprises non-human variable domains, wherein saidhumanized variant binds human VEGF and has those heavy chainComplementary Determining Region amino acid sequences as described andclaimed in the U.S. Pat. No. 6,884,879 which is hereby incorporated byreference. The anti-VEGF antibodies include those that can be producedusing vectors having nucleic acid encoding such CDR amino acid sequencesand isolated host cells containing such vectors. The host cells can becultured to produce such sequences and the humanized anti-VEGFantibodies may be recovered from such host cell cultures. The isolatednucleic acid recited above may further encode for a humanized varianthaving a light chain Complementary Determining Region (CDR) with thosesequences as recited in the '879 patent. Such humanized variant maycomprise a heavy chain variable domain having sequence shown as SEQ IDNO: 7 and a light chain variable domain having sequence shown as SEQ IDNO: 8 in the '879 patent. Such humanized variant may also comprise aheavy chain variable domain sequence of SEQ ID NO: 116 and a light chainvariable domain sequence of SEQ ID NO: 115 as shown in the '879 patent.

U.S. Pat. No. 7,060,269 is also incorporated by reference herein. Thispatent claims and discloses ranibizumab. Claim 1 of the '269 patentclaims a method for inhibiting VEGF-induces angiogenesis in a subject,comprising administering to said subject an effective amount of ahumanized anti-VEGF antibody which binds human VEGF with a Kd value ofno more than about 1×10⁻⁸, said humanized anti-VEGF antibody comprisinga heavy chain variable domain sequence of SEQ ID NO: 116 and a lightchain variable domain sequence of SEQ ID NO: 115. Ranibizumab is arecombinant humanized IgG1 kappa isotype monoclonal antibody fragmentdesigned for intraocular use. This monoclonal antibody binds to andinhibits human vascular endothelial growth factor A (VEGF-A).Ranibizumab has a molecular weight of about 48,000 daltons and isproduced in an E. coli expression system in a nutrient medium containingtetracycline. This product is commercially available under the tradenameLUCENTIS® and is supplied as a preservative free, sterile solution in asingle use glass vial that can deliver 0.05 mL of 10 mg/mL ranibizumabaqueous solution with 10 mM histidine HCl, 10% alpha, alpha trehalosedehydrate, 0.01% polysorbate 20, and at a pH of 5.5.

Ranibizumab is described in a scientific journal published in 1999 inthe Journal of Molecular Biology (JMB) by Chen et al. entitled“Selection and Analysis of an Optimized Anti-VEGF Antibody: CrystalStructure of an Affinity-matured Fab in complex with Antigen” ⁵(Chen etal. JMB, 293:865-881 (1999). The heavy chain and light chain sequencesof ranibizumab are designated as YO317 in this article and are shown inFIG. 1 therein. In addition to this description, the article alsoprovides data regarding binding affinity of this antibody fragment toVEGF (Table 6 on page 870 therein). Ranibizumab is known to bind to andinhibit the biological activity of VEGF-A which causesneovascularization and leakage in ocular angiogenesis models.Ranibizumab binds to and inhibits VEGF-A and prevents VEGF-A frominteracting with the VEGF receptors on the surface of endothelial cellsand thus reduces new blood vessel formation (angiogenesis); vascularleakage and endothelial cell proliferation. Administration of apharmaceutically effective amount of ranibizumab inhibits VEGF inducedangiogenesis. The term anti-VEGF antibody encompasses full lengthantibodies and antibody fragments such as Fab, Fab′, F(ab)₂ and F_(v)provided said fragments show the desired pharmacological activity bybinding to human VEGF. Ranibizumab has a binding affinity to VEGF (Kd)of no more than about 1×10⁻⁸ M-i.e., of about 1.4×10⁻¹⁰) (see Chen etal, on page 870). FIGS. 10A and 10B of the '269 patent provide thesequences of the light chain variable and heavy chain variable domainsof ranibizumab (Fab Y0317 as shown in Chen et al.). These sequences areidentical to SEQ ID NO: 115 and SEQ ID NO: 116 of the '269 patent.

In addition to ranibizumab and other anti-VEGF inhibitors or drugsdescribed in the above articles and patents, additional anti-VEGF oranti-angiogenic drugs may also be utilized in the present formulation.While the inventors have discovered that anti-VEGF antibodies that areantibody fragments are preferred in the liposomal formulations for thetreatment of diseases and conditions that involve topical application tothe eyes of patients having, healthy corneas or di minimusneovascularization of the cornea but some other ocular condition (e.g.age related macular degeneration or diabetic macular edema),formulations comprising whole anti-VEGF antibodies and self-forming,thermodynamically stable liposomes are also useful in the treatment ofconical neovascularization and these other ocular diseases (in patientshaving both diseases) provided the corneal neovascularization permits orfacilitates the entry of the large whole antibody and formulationsthereof. An example of an intact or whole anti-VEGF antibody is sold byGenentech under the brand name AVASTIN® (bevacizumab). This antibody isa recombinant humanized IgG1 antibody that inhibits the biologicalactivity of VEGF. It contains human framework regions and thecomplementarity-determining regions of a murine antibody that binds toVEGF and has an approximate molecular weight of 149 kD. This antibody isproduced in a mammalian cell (Chinese Hamster Ovary) expression systemin a nutrient medium containing Gentamycin U.S. Pat. No. 6,054,297(hereby incorporated by reference in its entirety) claims and disclosesbevacizumab or a process for making bevacizumab (see claims 1, 6, 7, 8,9, 10, 12, 29 and 30 therein).

As described in the approved product package insert, bevacizumab is arecombinant humanized monoclonal IgG1 antibody that binds to andinhibits the biological activity of human vascular endothelial growthfactor in in vitro and in vivo assay systems. This antibody containshuman framework regions of a murine antibody that binds to VEGF ⁶(see L.G. Presta et al. (1997) Cancer Res. 57: 4593-99). The molecular weightof bevacizumab is about 149 kilodaltons. This paper discloses theinteraction of the variable domains of the humanized F(ab) antibodyfragment, “F(ab)1-12.” Bevacizumab has non-human CDRs derived from thesequence of murine antibody as well as framework substitutions in thevariable domains at position 46 in the light chain (V_(L)) and positions49, 69, 71, 73, 78 and 94 in the heavy chain (V_(H)) that are the sameas the substitutions shown at the corresponding: positions of F(ab)-12,as shown in FIG. 1 of Presta et al. Presta et al. has information aboutthe molecular features and binding characteristics of bevacizumab.

As stated previously, In addition to ranibizumab and bevacizumab, otherknown anti-VEGF antibodies or anti-angiogenic drugs may also be utilizedin the present invention. The anti-VEGF antibodies of the invention areprepared as described in the patent references cited above with respectto same. In general, isolated nucleic acid encoding the antibody;vectors comprising the nucleic acid are operably linked to controlsequences recognized by host cells transformed with the vector; hostcells having said vector are all collectively used in a process forproducing the antibody of interest after culturing said cells andcollecting and purifying the antibody. Any suitable pharmaceuticalexcipient may be added to the antibody and the antibody may also belyophilized as desired. The “anti-VEGF antibodies” are inclusive ofvarious forms and may be full length having an intact human Fc region oran antibody fragment-e.g., Fab, Fab′ or F(ab′)₂. The otheranti-angiogenic drugs suitable for combining with the lipids disclosedherein to form liposomal formulations include pegaptanib or etanercept(a TNF inhibitor). In the latter case, this formulation may be used totreat various autoimmune diseases or conditions. Etauercept is soldunder the trade name Enbrel® which is used to treat rheumatoid, juvenilerheumatoid and psoriatic arthritis, plaque psoriasis and ankylosingspondylitis. Other suitable drugs include sunitinib, a VEGF and PDGFreceptor protein kinase and angiogenesis inhibitor (a 2-oxindole soldunder the name SUTENT®) and which is described and claimed in U.S. Pat.No. 6,573,293 hereby incorporated by reference) or FOVESTA™ (formerlyknown as E10030), a regulator of platelet derived growth factor B(PDGF-B)(1.5 mgs/0.5 mgs ranibizumab). Other suitable drugs used incombination include interferon-alpha-2a or temsirolimus or other mTORinhibitors such as rapamycin. Classes of drugs for ocular diseases thatmay he used in combination also include proteasomal inhibitors,autophagy inhibitors, retinoids, lysosomal inhibitors, heat shockresponse activators, Hsp90 chaperone inhibitors, protein transportinhibitors, glycosidase inhibitors, tyrosine kinase inhibitors andhistone deacetylase inhibitors. These drugs may be utilized alone in theliposomal formulation or may be used in a combination formulation withan anti-VEGF compound or antibody or may be used in sequentialcombination and preferably in a topical formulation. The preferredindication when combining FOVISTA™ (0.03-3.0 mgs/eye in combination with0.5 mg ranibizumab or other anti-VEGF compound)(and/or other drug havingPDGF inhibition activity) and ranibizumab is the treatment ofage-related macular degeneration. Aflibercept (2.0 mgs/0.05 mL)(Eylea™)may also be used in the liposomal formulation alone or in combinationwith ranibizumab or other active ingredients. The present inventionfurther includes a topical liposomal formulation as recited hereincomprising FOVISTA and aflibercept and/or any other anti-angiogenic drugprovided that at least one of the active ingredients is blended/combinedwith the thermodynamically stable, self forming liposomes of theinvention. FOVISTA (an aptamer directed against PDGF-B) is also knownand described as “Antagonist A” in U.S. Pat. Pub. No. 2012/0100136 whichis hereby incorporated by reference in its entirety. The synthesis ofAntagonist A is described in Example 4 in the '136 publication (see alsoFIG. 7 therein). Each of the individual VEGF antagonists and PDGFantagonists described therein are also included in the scope of thepresent invention when at least one agent is formulated with a lipiddescribed herein that forms a thermodynamically stable, self formingliposome. A topical formulation having any one of or any combination ofthe active ingredients recited herein is advantageous over the drugs orcombination thereof, that are administered by intravitreal injection. Inaddition, topical application to the eye for an ocular disease ispreferable to systemic oral administration. The compositions useful inthe present invention include, as liposomal formulations or liposomalformulations and/or any other formulation in combination, (a) (PDGFinhibitors) ARC-127, Antagonist A, Antagonist B, Antagonist C,Antagonist D, 1B3 antibody, CDP860, IMC-3G3, imatinib, 162.62 antibody,163.31 antibody, 169.14 antibody, 169.31 antibody, αR1 antibody, 2A1E2antibody, M4TS.11 antibody, M4TS.22 antibody, A10, brefeldin A,sunitinib, Hyb 120.1.2.1.2 antibody, Hyb 121.6.1.1.1 antibody, Hyb127.5.7.3.1 antibody, Hyb 127.8.2.2.2 antibody, Hyb 1.6.1 antibody, Hyb1.11.1 antibody, Hyb 1.17.1 antibody, Hyb 1.18.1 antibody, Hyb 1.19.1antibody, Hyb 1.23.1 antibody, Hyb 1.24 antibody, Hyb 1.25 antibody, Hyb1.29 antibody, Hyb 1.33 antibody, Hyb 1.38 antibody, Hyb 1.39 antibody,Hyb 1.40 antibody, Hyb 1.45 antibody, Hyb 1.46 antibody, Hyb 1.48antibody, Hyb 1.49 antibody, Hyb 1.51 antibody, Hyb 6.4.1 antibody, F3antibody, Humanized F3 antibody, C1 antibody, Humanized C1 antibody, 6.4antibody, anti-mPGDF-C goat IgG antibody, C3.1 antibody,5-methyl-7-diethylamino-s-triazolo (1,5-a) pyrimidine, interferon,protamine, PDGFR-B1 monoclonal antibody, PDGFR-B2 monoclonal antibody,6D11 monoclonal antibody, S is 1 monoclonal antibody, PR7212 monoclonalantibody, PR292 monoclonal antibody, HYB9610 monoclonoal antibody, HYB9611 monoclonal antibody, HYB 9612 monoclonal antibody, HYB 9613monoclonal antibody,4-(2-(N-(2-carboxamido-indole)aminoethyl)-benzenesulfonate,4-(2-(N-(-2-carboxamideindole)aminoethyl)-sulfonylurea, CGP 53716, humanantibody g162, pyrazolo[3,4-g]quinoxaline,6-[2-(methylcarbamoyl)phenylsulphanyl]-3-E-[2-(pyridine-2-yl)ethenyl]-indazole,1-{2-[5-(2-methoxy-ethoxy)-benzoimidazole-1-yl]-quinoline-8-yl}-piperidine-4-ylamine,4-4-[N-(4-nitrophenyl)carbamoyl]-1-piperazinyl]-6,7-dimethoxyquinazoline,4-amino-5-fluoro-3-(6-(4-methyl-piperazine-1-yl)-1H-benzimidazole-2-yl)1H-quinoline-2-one,(4-tert-butylphenyl){4-[(6,7-dimethoxy-4-quinolyl)oxy]phenyl}methancone,5-methyl-N-[4-(trifluoromethyl)phenyl]-4-izoxazolecarboxamide,trans-4-[(6,7-dimethoxyquinoxaline-2-yl)amino]cyclohexanol,(Z)-3-[(2,4-dimethyl-5-(2-oxo-1,2-dihydroindole-3-ylidenemethyl)-1H-pyrrole-3-yl)-propionicacid,5-(5-fluoro-2-oxo-1,2-dihydroindole-3-ylidenemethyl)-2,4-dimethyl-1H-pyrrole-3-carboxylicacid, 1-(4-chloroanilino)-4-(4-pyridylmethyl)phthalazine,N-{4-(3-amino-1H-indazole-4-yl)phenyl-N″-(2-fluoro-5-methylphenyl)urea,1,2-dimethyl-7-(2-thiophene)imidazole[5,4-g]quinoxaline,1,2-dimethyl-6-phenyl-imidazolo[5,4-g]quinoxaline,1,2-dimethyl-6-(2-thiophene)imidazolo[5,4-g]quinoxaline, AG1295, AG1296,3-arylquinoline, 4-pyridyl-2-arylpyrimidine, sorafenib, MLN518, PKC412,AMN107, suramin neomycin or a pharmaceutically acceptable salt thereofand (b) (VEGF inhibitors) ranibizumab, bevacizumab, aflibercept, KH902VEGF receptor-Fe fusion protein, 2C3 antibody, ORA102, pegaptanib,bevasiranib, blunt ended bevasiranib, SIRNA-027, deeursin, decursinol,picropodophyllin, guggulsterone, PLG101, eicosanoid LXA4, PTK787,pazopanib, axitinib, CDDO-Me, CDDO-Imm, shikonin,betahydroxvisovaleryishikonin, ganglioside GM3, DC101 antibody, Mab 25antibody, Mab73 antibody, 4A5 antibody, 4E10 antibody, 5F12 antibody,VA01 antibody, BL2 antibody, BECG-related protein, sFLT01, sFLT02,Peptide B3, TG100801, sorafenib, G6-31 antibody or other compounds whichinhibit VEGF related angiogenesis. In addition to antibodies, otherprotein based active ingredients suitable to treat ophthalmic diseasesor conditions, including diseases of the front of the eye such ascorneal diseases or healing necessary due to surgical incisions; traumaor ulcers, includes, for example, human growth hormones or other knownhormonal peptides or variants thereof.

The liposomal formulation is prepared by following the following generalsteps in any order: (1) provision of an aqueous solution containing ananti-VEGF antibody and/or other active or actives as described above;(2) addition of a thermodynamically stable, self-forming lipid capableof forming a liposome to said aqueous solution of step (1) and (3)optional addition of pharmaceutically acceptable excipients. Anyvariation of a process to prepare the liposomal suspension formulationmay be utilized including combining, the anti-VEGF antibody (or VEGFinhibitor or PDGF inhibitor) and the lipid and then adding an aqueoussolution or adding each ingredient separately to an aqueous solution.The suspension is prepared based upon the expected route of delivery(e.g. topical etc.) and the additional excipients are selected basedupon such route as well. Carriers, stabilizers and/or excipients includebuffers such as phosphate, citrate or other inorganic acids;antioxidants such as ascorbic acid and/or methionine; preservatives; lowmolecular weight polypeptides; proteins such as gelatin, serum albuminor immunoglobulins; hydrophilic polymers such as PVP; amino acids;monosaccharides or disaccharides or other carbohydrates; chelatingagents; sugars; salt forming counter-ions; non-ionic surfactants and thelike. The liposomal formulation may also be in the form of a solution.

The formulations are useful in the treatment of VEGF related diseasesand disorders. The preferred diseases or conditions to be treated withthe formulation described herein are ocular diseases. As describedabove, the preferred disease or condition for the present invention isthe treatment of diabetic macular edema. As referenced above, DMEresults from a series of biochemical and cellular events that ultimatelycause progressive leakage and exudation, leading to thickening of theretina and the formation of hard exudates within one disc diameter ofthe center of the macula. Laser photocoagulation is the mainstay oftreatment and is effective to prevent the risk of moderate visual lossby about 505 [ETDRSRG, 1985]. Laser photocoagulation leads toimprovement in reading line scores but has associated complication suchas progressive enlargement of scars, central scotomata, decreasedcontrast sensitivity and impaired color vision.

The liposomal formulation may broadly treat tumors or retinal disordersassociated with VEGF and/or PDGF or any other ophthalmic disease orcondition depending upon the particular active ingredient. The anti-VEGFantibodies inhibit one or more of the biological activities caused byVEGF. Therapeutic applications involve a pharmaceutically acceptabledosage form administered to a patient in need of treatment of theparticular disease or condition. Suitable dosage forms while preferablytopical may also include administration by intraveneous means as a bolusor by continuous infusion; intramuscular, intreperitoneal,intra-cerobrospinal, subcutaneous, intraarticular, intrasynovial,intrethecal, oral or by inhalation. In addition, such antibodyformulations may also be administered by intra tumoral, periturnoral,intrelesional or perilesional routes. The neoplastic diseases amenableto treatment with the antibody formulations include various carcinomasincluding breast carcinoma, lung carcinoma, gastric carcinoma,esophageal, colorectal, liver, ovarian, arrhenoblastomas, cervical,endometrial, endometrial hyperplasia, endometriosis, fibrosacromas,choriocarcinoma, head and neck cancer, nasopharyngeal carcinoma,laryngeal carbinomas, hepatolastoma Kaposi's sarcoma, melanoma, skincarcinomas, hemangioma, cavernous hemangioma, hemangioblastoma, pancreascarcinomas and other types of cancer. Non-neoplastic conditions that areVEGF related include rheumatoid arthritis, posriasis, atherosclerosis,diabetic and other proliferative retinopathies including retinopathy ofprematurity, retrolental fibroplasias, neovascular glaucoma, age-relatedmacular degeneration, diabetic macular edema and other forms of macularedema, thyroid hyperplasias including Grave's disease, corneal and othertissue transplantation, chronic inflammation, lung inflammation,nephritic syndrome, preecilampsia, ascites, pericardia effusions andpleural effusion. The preferred condition or disease treated with thepreferred topical formulation is diabetic macular edema. The dosageadministered and the frequency of administration will depend upon thetype and severity of the disease and the particular patient's condition.For example, the anti-VEGF antibodies may be administered at a dosagerange of 1 μg/kg to about 50 mg/kg or about 0.1-20 mg/kg to a patient inneed of treatment thereof. The preferred dosage regimen for thetreatment of DME and with respect to the topical formulation ofranibizumab is described in Example 3 herein. The concentration andamount of active ingredient may be varied depending upon the particularpatient and the number of days treated and amount provided per day orweek or month may also be varied depending upon the patient's responseand signs of improvement in both visual acuity and in retinalthickening.

An ideal treatment modality for purposes of treating DME or other VEGFrelated ocular condition would be one that leads to rapid and longlasting vision improvement. The other treatment modalities currentlyused for the treatment of DME include selective PKCβ inhibitors(ruboxistaurin); steroids (triamcinolone acetonide, fluocinoloneacetonide); VEGF inhibitors (bevacizumab; ranibizumab andpegaptinib-injectables) and vitrectomy. The present liposomalformulation provides a topical treatment regimen that is a significantimprovement over, for example, intravitreal formulations currently onthe market. The present formulation can be used in combination withother known treatments for the ocular or VEGF related diseases orconditions recited herein and/or as described above and provided thereare no contraindications. Such treatment regimens or therapeuticapproaches include, for example, siRNA molecules such as bevasiranib andwith appropriate delivery vehicles including the thermodynamicallystable, self forming liposomes utilized in the current invention.

Ophthalmic steroids that may be utilized in the liposomal formulationalone or in combination with any other active ingredient includedexamethasone, fluocinolone, loteprednol difluprednate, fluorometholone,prednisolone, medrysone, triamcinolone acetonide, rimexolone and thevarious salt forms thereof. Other ophthalmic anti-inflammatory agents(for example NSAIDs) may also be utilized in the liposomal formulation.Depending, upon the active ingredient, other liposomes in addition to oras an alternative to the thermodynamically stable self-forming liposomesmay be utilized.

The following examples are intended to further illustrate certainembodiments of the invention and are non-limiting:

EXAMPLES Example 1 A Solution of Liposomes and Ranibizumab

A vial containing 0.5 mg of ranibizumab at a concentration of 10 mg/mL(0.05 mL) was obtained. 0.015 grams of PEG-12 glycerol dimyristate(PEG-12 GDM) Qsomes™ was added to this solution (the number after PEGindicates the number of C₂H₄O subunits in the PEG chain). The volume ofthis liposomal suspension was diluted to a final volume of 1.5 mL using1.45 mL of a buffer solution consisting of phosphates, sodium chlorideand polioxyl 40 stearate to provide a ranibizumab concentration of 0.333mg/mL in the liposomal suspension and a lipid percentage of about 1% (10mg/mL). Sodium perborate (0.28 mg/mL) was added as a preservative. 1 mLof this suspension is equivalent to 20 drops. Each drop containsapproximately 17 μg ranibizumab.

The buffer solution was prepared by combining a 15 mL solution ofpolyoxyl 40 stearate, sodium chloride, sodium monobasic phosphate andsodium dibasic phosphate with 5 mLs of the sodium perborate solution(V=20 mL, pH 5.5). 1.45 mLs of this solution was then utilized asdescribed directly above. The concentration of each of the excipients inthe ophthalmic liposomal suspension formulation was 0.142 mg/mL (sodiumphosphate dibasic); 6.7 mg/mL (sodium phosphate monobasic); 50 mg/mL(polioxil 40 stearate); 5.1 mg/mL (sodium chloride); 0.333 mg/mL(ranibizumab); 10 mgs/mL (PEG-12 GDM) and 2.8 mg/mL (sodium perborate).The pH may be adjusted with HCl or NaOH and low molecular weight aminoacids or organic acids may be utilized as well. FIG. 3 shows at leasttwo types of liposomes (Qusomes®) that are formed when the lipid ismixed with an aqueous solution (from Biozone Laboratories website).

Example 2 Diffusion Chamber Study in Rabbit Corneas

Diffusion chamber data of the liposomal formulation applied to rabbitcorneas was generated using the methods described below. To summarize,samples were taken at 10, 20 and 30 minutes and at hours 1, 2, 3, 4, 5,6 and 24. The data showed a significant rate of penetration into theaqueous humor of rabbit corneas at 34 degrees Centigrade for theliposomal ranibizumab formulation applied topically In the liposomalformulation ranibizumab was identified starting at 3 hours and remainedpresent up to 24 hours post administration versus 7 and 14 dayspreviously reported in the rabbit for a non-liposomal formulation (datanot shown-see Chen et al., Eye London 2011 November; 25(11):1504-11.).Experiments were conducted in glass, Valia-Chen chambers with horizontalflow. The water recirculates with a temperature of 34 degrees C. Amembrane was placed between the junctions of the chambers and, in thisexample, rabitt corneas were used as the membrane. The receptor chamberwas filed with 3.2 mLs of saline solution to simulate the content ofaqueous humor in the anterior portion of the eye. The donor chamber wasprovided with 3 mLs of the ophthalmic formulation comprising ranibizumaband the thermodynamically stable, self-forming lipid. The diffusionchambers were constantly agitated. Samples were collected from thereceptor chamber at various timepoints-400 μL samples were taken andreplaced with 400 μLs of saline solution each time. The samples weretaken at time points: 10 min; 20 min; 30 min; 1 hr; 2 hr; 3 hr; 4 hr; 5hr; 6 hr and 24 hr. Ranibizumab was detected by HPLC as early as the3^(rd) hour. Lucentis® was used as a control solution for the HPLCstandard. Electrophorosis was also used to evaluate the passage of theliposomal formulation through the rabbit cornea membrane and resultswere consistent with the HPLC data.

Example 3 Pilot Clinical Study on Patients with DME

A patient having DME was treated six times/day with 1 drop/every threehours of the formulation on a daily basis (6×/day) for two weeks. Thetotal dose/day of ranibizumab was 6×17 ug or 102 ug. Improvements inloss of mean central foveal thickness (CFT) and an increase in visualacuity were seen through six weeks following this two week period (SeeFIG. 1 and FIG. 2). At week eight an increase in retina thickness anddecline in visual sharpness occurred and, at week 10, treatment wasreinitiated at two drops per day (34 ug/day). At week 14 clear tendencytoward improvement in OCT and BCVA was observed (see FIGS. 1 and 2). Twoadditional patients were also treated using the same protocol. Resultsof all three patients are presented in FIGS. 4-7 and show improvement inCFT and VA relative to control.

Example 4 Pilot Clinical Study on Patients with DME Using TriamcinoloneActetonide

Eligible patients having DME received a topical formulation comprisingtriamcinolone (TA) in a single center open label pilot study. A total of3 eyes of 3 patients (mean age 58 years, range 53-64) with DME involvingthe center of the macula and best-corrected visual acuity (BCVA) in thestudy eye between 65 and 40 letters using ETDRS testing. Patients wereinstructed to apply one drop containing 133 ug (micrograms) of TA everytwo hours in the study eye, while they were awake (six times) during thecontrolled treatment period of twelve (12) weeks. The main outcomemeasures included primary end points at three months such as thefrequency and severity of ocular and systemic adverse events and thechange from baseline in the central foveal thickness (CFT), as measuredby optical coherence tomography (OCT) over time. The secondary outcomeswere the change from baseline BCVA score over time, proportion ofpatients with a >3-step progression from baseline in ETDRS retinopathyseverity on fundus photographs (FP), proportion of patients withresolution of leakage on fluroescein angiography (FA) and the need ofmacular laser treatment over time. The TA formulation was prepared in asimilar manner to the ranibizumab formulation from commerciallyavailable starting materials.

Triamcinolone+1% Liposomes Ophthalmic Suspension

The final formulation is sterile, aqueous suspension. Its content is asfollows:

TA FORMULATION mg/mL Triamcinolone acetonide * 2.667Hydroxypropylmethylcellulose 3.000 Mono basic sodium phosphate 10.000Dibasic sodium phosphate 3.000 Polysorbate 80 0.500 EDTA 0.100 Sodiumchloride 2.500 Benzalkonium Chloride 50% 0.200 PEG-12-GDM 10.0000 Water1 mL NaOH or HCl to adjust pH 5.0-7.5 Each drop of the suspensioncontains 133.35 μg. PEG-12-GDM: Liposomes;Diacylglycerol-polyethyleneglycol (PEG 12), glycerol dimyristate(GDM). * TA is a finished product. It is TA micronized (approx. 12 mm)and free of preservatives.

Preparation Protocol of Triamcinolone+1% Liposomes Ophthalmic Suspension

-   1. Place 40% of the final volume of distilled water in a beaker and    heat it to 70 to 80° C.-   2. Add the hydroxypropylmethylcellulose and stop mixing until    reaching room temperature and it becomes a clear and homogeneous    mixture.-   3. Autoclave it and once sterile allows it to reach room temperature    while stirring.-   4. Place in another beaker 40% of the final Volume of distilled    water. Add and mix until completely dissolved one by one the    following reagents:    -   a) Sodium phosphate monobasic    -   b) Sodium phosphate dibasic    -   c) EDTA    -   d) Sodium chloride    -   e) Polysorbate 80-   5. In 10% of the remaining volume of water, add the benzalkonium    chloride at 50% and mix until completely incorporated. Once    dissolved, add this new solution to the above solution containing    phosphates, EDTA, sodium chloride and Polysorbate 80. To sterilize,    filter by 0.22 μm membrane.-   6. Mix the sterile solution of hydroxypropylmethylcellulose with the    other sterile solution containing the salts and the preservative    benzalkonium chloride and mix until getting a clear homogeneous    mixture.-   7. Add the triamcinolone acetate to the solution with buffers and    benzalkonium chloride and stir until completely incorporated.-   8. Add the Liposomes to this mix and stir during 15 minutes with a    magnetic stirrer to obtain a final suspension.-   9. Package the suspension in special eye dropper. Each dropper    bottle contained 1.5 mL of this triamcinolone ophthalmic suspension.

Results: The use of a topical formulation comprising TA in the liposomalformulation in patients with center-involving clinically significant DMEwas well tolerated. Neither ocular nor systemic adverse events werereported. At month 2, the CFT of all three patients was reduced relativeto baseline. Two of the three patients had a decrease in CFT of at least100 um. At month three, all three patients showed visual acuityimprovement. One of the patients gained >15 letters.

While the claimed invention has been described in detail and withreference to specific embodiments thereof, it will be apparent to one ofordinary skill in the art that various changes and modifications can bemade to the claimed invention without departing from the spirit andscope thereof. Thus, for example, those skilled in the art willrecognize or be able to ascertain using no more than routineexperimentation, numerous embodiments of the claimed invention which maynot have been expressly described. Such embodiments are within the scopeof the invention.

REFERENCES

-   1. Roskoski Jr. Sunitinib: A VEGF and PDGF receptor protein kinase    and angiogenesis inhibitor. BBRC, 2007.-   2. Fong D, Diabetic Retinopathy. Diabetes Care, 2004.-   3. Bhagat N, Diabetic Macular Edema: Pathogenesis and Treatment.    Survey of Ophthalmology 2009.-   4. Meyer C, Current Treatment Approaches in Diabetic Macular Edema.    Ophthalmologica, 2007.-   5. Chen Y, Selection and analysis of an optimized Anti-VEGF antibody    Crystal structure of an affinity-matured Fab in complex with    antigen, JMB, 1999.-   6. Presta L, Humanization of an Anti-vascular endothelial growth    factor monoclonal antibody for the therapy of solid tumors and other    disorders.-   7. ETDRSR, Photocoagulation for macular Edema, Report 1, 1985,

What is claimed is:
 1. A topical aqueous ophthalmic formulation fortreating a posterior segment disease or condition by topical delivery tothe eye comprising a non-ionic surfactant, a pharmaceutically effectiveamount of a steroid and a thermodynamically stable, self-formingliposome formed from a PEG-based lipid wherein the weight percentage ofthe lipid in the total weight of the formulation is less than 20% wt/wtwherein the steroid is selected from triamcinolone acetonide; theliposome is selected from the group consisting of a PEG-12 glyceryldimyristate or a PEG-12 glyceryl dioleate and the pH of the topicalaqueous ophthalmic formulation ranges from about 5.0 to 7.5.
 2. Theformulation according to claim 1 wherein the posterior segment diseaseor condition is selected from age-related macular degeneration ordiabetic retinopathy.
 3. A topical ophthalmic formulation comprising athermodynamically stable self-forming liposome having a polyethyleneglycol (PEG) chain and a glycerol backbone, an active ingredientselected from triamcinolone acetonide, a surfactant, and a bufferreagent which maintains the pH at a range of about 5 to 7.5 wherein theglycerol backbone is selected from the group consisting of glyceroldilaurate (GDL), glycerol dioleate (GDO), glycerol dimyristate (GDM),glycerol dipalmitate (GDP) or glycerol distearate (GDS) and the PEGchain is selected from PEG-12 and the buffer reagent is a citrate bufferand the surfactant is a non-ionic surfactant and wherein the liposome isa self-forming thermodynamically stable liposome present in a weightpercentage of less than 20% wt/wt based upon the total weight of theformulation.
 4. The formulation according to claim 3 wherein the thethermodynamically stable self-forming liposome is selected fromPeG-12-GDM, PEG-12-N1-GDO, PEG-12-Ac2-GDO; PEG-12N1-GDM; orPEG-12-Ac2-GDM; wherein N1 is amine; Ac1 is succinyl and Ac2 is acetyl.5. A method of treating diabetic macular edema in a patient in need oftreatment thereof comprising topically administering to the surface ofthe eye a pharmaceutically effective amount of a topical ophthalmicformulation comprising a thermodynamically stable self-forming liposomeselected from the group consisting of PEG-12-GDM or PEG-12 GDO andtriamcinolone acetonide wherein the liposome is a self-formingthermodynamically stable liposome present in a weight percentage of lessthan 20% wt/wt based upon the total weight of the formulation.
 6. Themethod according to claim 5 wherein triamcinolone acetonide isadministered at a dosage range of 50-150 μg/per drop of formulation andapplied topically one drop to the eye every two hours 4-6×/day.
 7. Themethod according to claim 5 wherein the patients showed an improvementin central foveal thickness and/or visual acuity.
 8. A method oftreating a patient having diabetic retinopathy or diabetic macular edemacomprising administering a liposomal topical ophthalmic formulationcomprising a self-forming thermodynamically stable liposome comprising aPEG-based lipid conjugate selected from a diacylglycerol-PEG compoundwherein the conjugate has a melting point below about 40 degrees C; anacyl chain of greater than or equal to about 14 carbons in length andthe PEG chain has a molecular weight of between about 300-5000 Daltonsand an active pharmaceutical agent selected from triamcinolone acetonidewherein said formulation is suitable for topical delivery to the eye ofthe patient to treat the posterior segment disease or condition withoutside effects or complications associated with an intravitreal injectionwherein the posterior segment disease or condition is selected fromdiabetic retinopathy or diabetic macular edema and wherein the weightpercentage of the self-forming thermodynamically stable liposome (wt/wt)is less than 20% based upon the total weight of the formulation.
 9. Themethod according to claim 8 wherein the side effects or complicationsare selected from the group consisting of patient discomfort, infectiousendophthalmitis, retinal detachment, intraocular pressure changes andtraumatic cataract.
 10. The method according to claim 8 wherein theophthalmic formulation further comprises a surfactant and a bufferreagent.
 11. The method according to claim 10 wherein the surfactantcomprises a non-ionic surfactant triamicinolone acetonide.
 12. Themethod according to claim 11 wherein the corticosteroid is selected fromtriamicinolone acetonide and the self forming thermodynamically stableliposome comprises PEG-12-glyceryl dimyristate or PEG-12 glyceryldioleate.
 13. The method according to claim 12 wherein the self-formingthermodynamically stable liposome is selected from PEG-12-gylceryldimyristate.
 14. The method according to claim 9 wherein the activeingredient is triamcinolone acetonide; the self-formingthermodynamically stable liposome is PEG-12-GDM and the surfactant is anon-ionic surfactant.
 15. The method according to claim 8 wherein thethermodynamically stable self-forming liposome is liquid at 25 degreesC., is self-forming at both 20 degrees C. and at 37 degrees C. and formswhen combined with aqueous solution.
 16. The method according to claim 8with the proviso that dexamethasone is excluded.